Separation method of macrophage in thyroid papillary carcinoma tissue

A technology of papillary carcinoma and macrophages, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve problems such as poor prognosis, increased mortality, and lost opportunities for surgery

Inactive Publication Date: 2013-10-23
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Although the mortality rate of PTC is relatively low compared with other malignant tumors, the metastasis rate of PTC is very high, and the rate of lymph

Method used

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  • Separation method of macrophage in thyroid papillary carcinoma tissue
  • Separation method of macrophage in thyroid papillary carcinoma tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1, Collection of Papillary Thyroid Carcinoma Specimens

[0027] Surgical specimens of papillary thyroid carcinoma (PTC) will be sent to the pathology department for frozen section pathological diagnosis within half an hour after surgical resection of thyroid tissue by staff in the operating room. Papillary thyroid cancer focus tissue will not affect the follow-up and complete pathological diagnosis. The pathologist will guide and help to collect fresh surgical tissue specimens of papillary thyroid cancer. Fetal calf serum and D-Hank's solution of antibiotics were placed in 50mL sterile centrifuge tubes and immediately sent to the laboratory for further testing experiments.

Embodiment 2

[0028] Example 2. Isolation process of macrophages in papillary thyroid carcinoma tissue

[0029] (1) Transfer the papillary thyroid carcinoma specimen in Example 1 to a 10 cm petri dish filled with D-hanks solution, put an ice box under the petri dish, and wash the tumor tissue 3 times.

[0030] (2) Discard most of the D-hanks solution, retain a small amount of D-hanks to keep the tissue moist, use ophthalmic surgical scissors to carefully cut off the connective tissue around the papillary thyroid carcinoma, and cut the tissue into 1×1×1mm 3 The size of the organization block.

[0031] (3) Transfer the shredded tissue particles to a sterile 50mL centrifuge tube, add type II collagenase to make the final concentration of the shredded tissue particles reach 5mg / mL (the volume of type II collagenase is 5 times the volume of the tissue) About), as for 2 hours of shaking digestion in a 37°C water bath.

[0032] (4) During the digestion process, observe the digestion progress of ...

Embodiment 3

[0039] Validation of Isolated Papillary Thyroid Carcinoma-Associated Macrophages:

[0040] The macrophages in the papillary thyroid carcinoma isolated in Example 2 were subjected to CD68 immunofluorescence staining, and the results were as follows figure 1 As shown, the isolated macrophages are round and can be tightly adhered to the culture dish. The CD68 staining is positive, and the positive rate is as high as 95%, which confirms the successful isolation of macrophages. Thyroglobulin (Tg) staining was performed at the same time, see figure 2 As shown, the staining was negative, ruling out contamination by thyroid follicular epithelial cells.

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Abstract

The invention provides a cell separation technology, and particularly relates to a separation method of macrophage in a thyroid papillary carcinoma tissue. The separation method comprises the following separation steps of: placing the collected thyroid papillary carcinoma tissue into D-hanks liquid; flushing; removing D-hanks liquid and connective tissues; cutting the collected thyroid papillary carcinoma tissue into blocks; adding II type collagenase into the blocked tissue; performing digestion reaction for 1 to 3 hours at temperature of 35 to 40 DEG C; adding an 1640 culture medium containing 10% of fetal calf serum for stopping the digestion reaction; filtering; centrifuging; removing supernatant; resuspending and planking cell sap through the 1640 culture medium; transferring into a cell incubator containing 5% of CO2 for incubating; fully washing through PBS (Phosphate Buffer Solution); re-culturing the obtained anchorage-dependent cells; collecting the supernatant; and freezing and storing. According to the separation method, the separated macrophage is circular and can be tightly adhered to a petri dish and shows positive by staining by CD68; the positive rate reaches more than 95%, namely, the macrophage is separated successfully.

Description

technical field [0001] The invention relates to a cell separation technology, in particular to a method for separating macrophages in thyroid papillary carcinoma tissue. Background technique [0002] The tumor microenvironment refers to the general term for all cells other than tumor parenchymal cells, including resident cells such as fibroblasts at the tumor site, and cells that migrate and infiltrate to the local area such as lymphocytes and macrophages, among which the largest number is Macrophages that migrate to the tumor site through blood transport are the main components of the tumor microenvironment. Macrophages are derived from bone marrow CD34+ progenitor cells. They first proliferate and divide in the bone marrow, then migrate to different tissues in the form of monocytes through blood transport, and differentiate into macrophages in different environments. Under physiological conditions, macrophages are involved in different life activities, including inflammat...

Claims

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Application Information

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IPC IPC(8): C12N5/0786
Inventor 宁光方微园李小英叶蕾
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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